Mouse anti vinculin antibody

For immunocytochemistry, cells were plated on a glass‐bottom chamber slide (Matsunami Glass, SCS‐008, Osaka, Japan). After fixation, cells were washed, and permeabilized with 0.5% Triton X‐100. Subsequently, cells were washed, and incubated with blocking buffer that contained 5% goat serum and 1% BSA. After blocking, cells were incubated with the primary antibodies, mouse anti‐vimentin antibody (Sigma, V6389, St. Louis, MO, 1:20 dilution), mouse anti‐E‐cadherin antibody (BD Biosciences, 610182, San Jose, CA, 1:50 dilution), rabbit anti‐phospho‐paxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, 1:20 dilution) and mouse anti‐vinculin antibody (Sigma, V9131, 1:200 dilution). Cells were washed, and stained with the secondary antibodies, rabbit anti‐mouse IgG antibody conjugated to Alexa 568 (Life Technologies, A11061, Carlsbad, CA, 1:200 dilution), goat anti‐rabbit IgG antibody conjugated to Alexa Fluor 546 (Life Technologies, A11010, 1:1000 dilution) and goat anti‐mouse IgG conjugated to Alexa Fluor 488 (Life Technologies, A11001, 1:1000 dilution). In cells immunostained for vimentin and E‐cadherin, nucleus was counterstained with DAPI. Fluorescent images were obtained using an all‐in‐one microscope (Keyence, BZ‐9000, Osaka, Japan).

iPSC-CMs or HEK293Ts were resuspended in lysis buffer (10% sodium dodecyl sulfate, 62.5 mM Tris [pH 6.8], 1 mM EDTA, and protease inhibitor). Mouse tissues were flash-frozen and crushed into a fine powder before being resuspended in lysis buffer. Protein concentration was determined by BCA assay, and 20–50 μg of total protein was loaded onto a 4%–20% acrylamide gel. Blots were then incubated with anti-dystrophin antibody (Sigma-Aldrich; D8168, 1:1000), anti-Cas9-N-terminal (Cell Signaling Technology; 7A9-3A3, 1:500), or anti-Cas9-C-terminal (Sigma Aldrich; 10C11-A12, 1:500) at 4°C overnight or with mouse anti-vinculin antibody (Sigma-Aldrich; V9131, 1:1,000) at room temperature for 1 h, followed by horseradish peroxidase antibody (Bio-Rad Laboratories) at room temperature for 1 h. Blots were developed using western blotting luminol reagent (Santa Cruz Biotechnology; sc-2048). Relative protein expression (densitometry) was measured using ImageJ’s Gel Analysis method, normalized to vinculin expression, and compared with the normalized WT dystrophin protein expression.